Screening of lactic acid bacteria with anti-adipogenic effect and potential probiotic properties from grains

A total of 187 lactic acid bacteria were isolated from four types of grains collected in South Korea. The bacterial strains were assigned as members of Levilactobacillus brevis, Latilactobacillus curvatus, Lactiplantibacillus plantarum, Lactococcus taiwanensis, Pediococcus pentosaceus, and Weissella paramesenteroides based on the closest similarity using 16S rRNA gene sequence analysis. The strains belonging to the same species were analyzed using RAPD-PCR, and one or two among strains showing the same band pattern were selected. Finally, 25 representative strains were selected for further functional study. Inhibitory effects of lipid accumulation were observed in the strains tested. Pediococcus pentosaceus K28, Levilactobacillus brevis RP21 and Lactiplantibacillus plantarum RP12 significantly reduced lipid accumulation and did not show cytotoxicity in C3H10T1/2 cells at treatment of 1–200 μg/mL. The three LAB strains decreased significantly expression of six adipogenic marker genes, PPARγ, C/EBPα, CD36, LPL, FAS and ACC, in C3H10T1/2 adipocytes. The three strains survived under strong acidity and bile salt conditions. The three strains showed adhesion to Caco-2 cells similar to a reference strain LGG. The resistance of the three strains to several antibiotics was also assessed. Strains RP12 and K28 were confirmed not to produce harmful enzymes based on API ZYM kit results. Based on these results, strains K28, RP21 and RP12 isolated from grains had the ability to inhibit adipogenesis in adipocytes and potentially be useful as probiotics.

The objectives of the present study were to isolate LAB from various grains collected in the Republic of Korea and to screen lactic acid bacteria with inhibition of adipogenesis. The selected strains with anti-adipogenic effects were investigated to determine whether they have useful properties as probiotic candidates for development as functional food.

Material and methods
Isolation of LAB strains and growth conditions. Four types of grains, rice, brown rice, black rice and hulled barley, were collected in the Republic of Korea. The grains were ground using blender and enriched for 7 days with distilled water under aerobic conditions. The grain samples were serially diluted with 0.85% (w/v) saline solution and spread on De Man-Rogosa-Sharpe (MRS; BD Difco, Sparks, MD, USA) agar. After incubation for 48 h at 30 ℃ or 37 ℃, LAB were isolated from the MRS agar plates and cultivated aerobically for 18 h at 30 ℃ or 37 °C in MRS agar. Lacticaseibacillus rhamnosus LGG (KCTC 5033), which was used as the experimental control strain for comparative analyses, was purchased from the Korean Collection for Type Cultures (Daejeon, Republic of Korea), and cultured at 37 ℃. All strains, including the experimental control strain, were stored at − 80 ℃ after suspension in 20% (w/v) glycerol solution (Georgiachem, GA, USA).
16S rRNA gene sequence analysis and random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) analysis. Genomic DNA was extracted using a G-spin genomic extraction kit (iNtRON, Seongnam, Republic of Korea), according to the manufacturer's protocol. Polymerase chain reaction (PCR) amplification, purification, and sequencing of 16S rRNA gene were performed as described previously 20 . Identification of the closest phylogenetic species based on 16S rRNA gene sequence was performed using the EzBioCloud server (https:// www. ezbio cloud. net/) 21 .
Preparation of cell extract from LAB. Cell extracts of LAB were prepared as described previously 23 with minor modifications. The cell mass was harvested using centrifugation and washed twice with phosphatebuffered saline (PBS, pH 7.2). The washed cells were resuspended in distilled water at a concentration of 100 mg/ mL and sonicated using the method described previously 16 . The sonicated cell extracts were centrifuged at 13,000 rpm for 15 min at 4 °C and the supernatants were filtered using a 0.45 µM syringe filter (Sartorius Stedim Biotech GmbH, Göttingen, Germany) and lyophilized. The resulting powder was dissolved in sterile water to appropriate concentrations.
Cell extracts of LAB were adjusted by suspending to a concentration of 25, 50, and100 μg/mL with sterile distilled water to create the same conditions. LAB cell extracts were treated during adipocyte differentiation of C3H10T1/2 cells, and sterile distilled water was treated as control. Then, the differentiated C3H10T1/2 cells were fixed with 4% formaldehyde (Sigma-Aldrich) in PBS (Hyclone) at room temperature overnight and stained with Oil Red O (Sigma-Aldrich). To quantify intracellular triglyceride content, stained cells from at least two independent experiments were resolved in isopropanol (Sigma-Aldrich) and measured with a spectrophotometer at 520 nm.
Quantitative real-time polymerase chain reaction (RT PCR) analysis. Total RNA was extracted from C3H10T1/2 cells using QIAzol lysis reagent (QIAGEN, Germantown, MD, USA). First-strand complementary DNA was synthesized from 0.5 μg of total RNA using ReverTra Ace Master Mix (Toyobo, Osaka, Japan) according to the manufacturer's instructions. Quantitative RT PCR was performed in 25 μL final reaction volume containing Power SYBR Premix ExTaq (RP041A; Takara, Shiga, Japan), primers, and cDNA using thermal cycler machine (Takara). The primer sequences used for the PCR were described previously 24  In vitro adhesion assays. The adherence assay was performed according to the method described previously 27 with minor modifications. Caco-2 cells used for the adherence assay were purchased from the Korean Cell Line Bank (Seoul, Korea). The Caco-2 cells were cultured in high glucose DMEM supplemented with 10% (v/v) FBS (Hyclone) and 1% (v/v) penicillin-streptomycin at 37 °C in 5% CO 2 atmosphere. The Caco-2 cells were seeded at 2 × 10 5 cells/well in 6-well tissue culture plates. The adherence assay was performed at post-confluence. The monolayer was washed with sterile PBS (Hyclone) twice. LAB cells were diluted with DMEM to approximately 10 9 CFU/mL and added to the wells. Plates were incubated for 90 min at 37 °C in 5% CO 2 atmosphere. The Caco-2 monolayers were washed three times with sterile PBS (Hyclone) and treated with EDTA-trypsin solution for 3 min. The cell suspensions were serially diluted and spread on MRS agar plates. Cell viability was counted after incubation for 48 h. The adhesion ability of LAB was calculated as the percentage between remaining bacteria and initial bacteria per well. The same passage Caco-2 cells were used in adhesion assays and assays were repeated in triplicate.
Antibiotic susceptibility. Susceptibility to antibiotics was examined using the disc-diffusion method with application of modified agar diffusion method described previously 28,29 . LAB inoculated in MRS agar were adjusted to approximately 10 8 CFU/mL and paper discs (Advantec, Tokyo, Japan) were dispensed. Each disc was treated with 10 μL of specific antibiotic. Enzyme activity test. Enzyme activity of the LAB was investigated as described previously 16 using the API ZYM kit (BioMérieux, Marcy l'Etoile, France).

Statistical analysis.
Results are presented as mean ± standard error of the mean (SEM) of three independent experiments. Significance differences between groups in triglyceride content were determined using Duncan's multi-range test. Significance differences in gene expression and adhesion ability were determined by comparison with control using two-tailed unpaired Student's t-test. A p-value < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS Inc. software (version 19.0).

Results and discussion
Isolation and identification of LAB strains from grains. Bacterial  In RAPD-PCR analysis, six different band patterns were assigned to 82 strains with the closest 16S rRNA gene sequence similarities to Pediococcus pentosaceus, and four different band patterns were assigned to 43 strains with the closest 16S rRNA gene sequence similarities to Weissella paramesenteroides ( Supplementary Fig. 1). The strains assigned as Levilactobacillus brevis, Latilactobacillus curvatus, Lactiplantibacillus plantarum, and Lactococcus taiwanensis each showed only one type of band pattern ( Supplementary Fig. 1). Finally, two representative strains from each group, except for the three groups having only one strain, were randomly selected, and used for further functional characterization (Supplementary Fig. 1; Table 1). www.nature.com/scientificreports/ plantarum RP11 and RP12) reduced lipid accumulation by more than 20% compared with the control, indicating that they have anti-adipogenic effects (Fig. 1). The two strains (RP20 and RP21) assigned to Levilactobacillus brevis and the two strains (RP11 and RP12) of Lactiplantibacillus plantarum showed similar results, respectively (Fig. 1). Thus, one strain from RP20 and RP21 and one strain from RP11 and RP12 were selected, and the three strains (K28, RP21 and RP12) were used for further experiments. The above results indicate that the components of LAB cell extract might influence the adipocyte differentiation process, thereby suppressing fat production. Significant diversity exists among LAB strains regarding functional characteristics that benefit health, such as antioxidant, antitumor, immunomodulatory, and hypocholesterolemic activities 34-40 . In several studies, the cellular components of LAB have shown beneficial effects on improving health 38,41,42 . It is not clear which substance(s) in LAB cell extract induce the anti-adipogenic effects. Exopolysaccharide (EPS) has been known to have antiadipogenic effects 23 . The EPS, a cell wall component of LAB cells, is loosely associated with the cell envelope and easily released into the surrounding environment 43,44 . Effects of LAB strains on cell viability of C3H10T1/2. The cytotoxicity at various concentrations of strains LGG, K28, RP21, and RP12 on C3H10T1/2 cells was investigated by measuring cell viability using the  www.nature.com/scientificreports/ MTT assay. C3H10T1/2 cells were found viable at all treatment concentrations of the four strains (Fig. 2). These results indicate that strains LGG, K28, RP21, and RP12 cause no damage to C3H10T1/2 cells 45 .

Inhibition of adipogenic gene expression by LAB extract during adipocyte differentiation.
Inhibition of adipogenesis by strains K28, RP21 and RP12 was investigated by measuring expression of six adipogenic genes using quantitative RT PCR (Fig. 3). PPARγ and C/EBPα are transcription factors that regulate the process of adipocyte differentiation 46,47 . In addition, the activation of PPARγ promotes the expression of adipogenic genes, such as CD36 and LPL, which are important for the uptake and storage of triglycerides 48 . The down regulation of these adipogenic genes may affect decreased lipid accumulation in cells. Fatty acid synthase (FAS) gene is a downstream adipocyte gene that contributes to fatty acid synthesis 49 . Acetyl-coenzyme A carboxylase (ACC ) is another key enzyme for fatty acid synthesis that catalyzes the synthesis of malonyl-CoA 50 . Cell extracts from strains K28, RP21, and RP12 decreased the expression of adipocyte-related genes in C3H10T1/2 cells (Fig. 3). The expression of the six genes decreased proportionally with increasing concentrations of the extracts of the three strains (Fig. 3). The three strains significantly reduced (p < 0.01 or 0.001) the expression of PPARγ and C/EBPα in all concentrations tested. In addition, expression of four other genes associated with adipogenesis, was significantly reduced (p < 0.05 or 0.01) in the three strains, except for LPL expression in 25 μg/ mL treatment of strains K28 and RP12. Strain K28, which showed the lowest lipid accumulation, was analyzed to have the lowest values in expressions of the six genes after 100 μg/mL treatment (Fig. 3). These results indicate that the three strains may have anti-adipogenic effects by inhibiting the expression of adipogenesis-related genes.
Tolerance against acid and bile salts. To have specific functionality, a probiotic must reach the intestines alive with resistance to acid and bile salts 51 . The acid tolerance of the selected strains and LGG as a reference strain was examined after incubation for 3 h in pH 3.0, 2.5, 2.0, and 1.0 ( Table 2). The three strains and LGG maintained the values of more than 9 log CFU/mL at pH 3. Under pH 2.5 condition, the survival rates of strains K28 and RP21 decreased more than 2 log and approximately 1 log, respectively, whereas strains RP12 and LGG showed decreases of more than 3 log and 2 log, respectively ( Table 2) www.nature.com/scientificreports/ of strains K28 and RP21 decreased approximately 3 log and 2 log, respectively, and the survival rate of strains RP12 and LGG decreased approximately 6 log ( Table 2). The three strains, except RP21 with approximately 3 log CFU/mL, showed low viability of less than 2 log CFU/mL at pH 1 ( Table 2). Strain RP21 was also found to have higher acid resistance, as a strain of Levilactobacillus (Lactobacillus) brevis was shown highly acid-resistant in a previous study 52 . The pH of gastric fluid in the body is maintained at approximately 3.0, and probiotics are generally known to be highly acid-resistant if they are maintained at pH 3 for approximately 3 h 53 . Thus, the three strains were concluded to be highly tolerant to acid. Because food matrix can help the survival of LAB in the gastrointestinal tract due to its buffering capacity, the strains are expected to have stronger viability when used with carrier foods 54 . Bile salts are another factor that can reduce bacterial survival in the gastrointestinal tract by destroying cell membranes 51 . Strains K28, RP21 and RP12 were found to survive after 6 h exposure to 0.3, 0.5, 1.0, and 2.0%  www.nature.com/scientificreports/ bile salts, similar to LGG which is known to be highly resistant to bile salts ( Table 2). Although the in vitro assay cannot provide the same conditions as the gastrointestinal tract, it is recognized as an effective evaluation method to select potential strains when using proper criteria 27 . Adherence to Caco-2 cells. The adhesion ability of probiotics is a main factor that can increase the possibility of their survival and colonization in the gastrointestinal tract 55 . Adhesion is also required to prevent attachment of pathogenic bacteria through competition in intestinal epithelium 56 . Thus, the adherence ability has been considered an important biological property for the selection of useful probiotic strains 57 . In the present study, the adhesion ability of the three strains was evaluated using Caco-2 cells, which have morphological and physiological properties of human enterocytes, and their adhesion abilities were compared with that of the reference strain LGG (Fig. 4). Strain K28 had stronger adhesion ability than those of LGG and the two other strains (Fig. 4). The adhesion ability of strain K28 was highest at 1.95%, followed by LGG (1.79%), RP12 (1.67%), and RP21 (1.46%).
Antibiotic susceptibility. Probiotics 59,60 . Strains K28, RP21, RP12 and LGG were resistant to gentamycin, kanamycin, and streptomycin, which are known to inhibit protein synthesis targeting Gram-negative bacteria. The resistance to aminoglycoside antibiotics is an intrinsic property among Lactobacillus species and Pediococcus species 61 . Therefore, the three strains are unlikely to cause safety problems based on antibiotic susceptibility profile tested.
Enzyme production. For the safety of probiotic strains, it may be required to assess whether the strains produce harmful enzyme. β-glucuronidase is known as the carcinogen enzyme, which may increase the likelihood of tumor induction in the colon 62,63 . When the three strains were evaluated using API ZYM kit, strains RP12 and K28 did not produce any harmful enzymes such as β-glucuronidase, but strain RP21 was observed to produce β-glucuronidase (Supplementary Table 2).

Conclusion
In the present study, 187 LAB strains were isolated from four types of grains and identified using 16S rRNA gene sequence analysis. The 25 strains selected based on RAPD-PCR analysis were subjected to functional characterization. Among the strains tested, Pediococcus pentosaceus K28, Levilactobacillus brevis RP21, and Lactiplantibacillus plantarum RP12 had the potential to be useful probiotic candidates based on several characteristic analyses. The three strains exerted inhibitory effects on lipid accumulation and adipocyte differentiation by decreasing the expression of adipocyte-related genes. In addition, the three strains showed good tolerance against acid and bile salts, good intestinal cell adhesion, and were sensitive to chloramphenicol and rifampicin. In particular, strains RP12 and K28 did not produce β-glucuronidase. Therefore, Pediococcus pentosaceus K28, Levilactobacillus brevis www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.